Molecular organization and structural stability of .beta.s-crystallin from calf lens

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Abstract
.beta.s-Crystallin has been purified to homogeneity. Its structural features and conformational behavior have been studied in solution. Protein secondary structure was estimated by curve fitting of far.sbd.UV circular dichroism spectra, which gave 16% .alpha.-helix, 45% .beta.-sheet, 12% bends, and 27% remainders. This result indicates that the structural organization of .beta.s-crystallin is reasonably similar to that of other .beta. and .gamma. family members. A comparison assessed between .beta.s- and .gamma.2-crystallin by the use of predictive methods (flexibility and volume plots) reveals that the two proteins differ in respect to their local flexibility and packing, although they show similar overall organization. The interdomain and the C-terminal regions were found to be more flexible in .beta.s-crystallin. This finding can be explained by the presence of smaller amino acid residues within these structural districts. The location of one out of four tryptophans, i.e., Trp-162, in a flexible and exposed region of the protein was found to be the origin of the fluorescence heterogeneity. In fact, the fluorescence emission maximum of the native protein, centered at 328 nm, is due to two emitting centers, whose emission maxima are located at 323 and 330 nm, respectively, as evidenced by acrylamide quenching of fluorescence. The effect of perturbing agents, such as pH and guanidine hydrochloride, on the conformational behavior of .beta.s has also been evaluated by numerous spectroscopic techniques. The range of pH stability was between 6.5 and 8. Above this interval, a conformational change takes place. In the acid region, the protein is unstable and precipitates irreversibly. The conformational resistance to guanidine hydrochloride has also been shown to be weak. GdnHCl denaturation curves were neither superimposable nor ascribable to a very cooperative transition. This result suggests a non-two-state denaturation equilibrium reflecting the presence of structural domains. The main conclusion of our work is that the protein shows a very narrow range of stability. This result indicates that an inherent structural stability may not be a general property of lens proteins. Therefore, the evolutive hypothesis about a specific recruitment of stable proteins in the lens architecture may need reconsideration.